JP2008169131A - Dna vaccine against crustacean acute viraemia - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a vaccine effective for prophylaxis of crustacean acute viraemia. <P>SOLUTION: The DNA vaccine for the prophylaxis of crustacean acute viraemia comprises a pTargeT (R) vector containing a structural protein gene of a WSSV (white spot syndrome virus). <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a DNA vaccine effective for preventing viral infection in crustaceans.

  Crustacean acute viremia (also called white spot syndrome, vitiligo disease, etc.) is a viral disease that has been reported the most serious damage in crustacean aquaculture. This disease is highly lethal within a few days after infection, and no effective prevention / treatment method has been established. Therefore, the current effective countermeasure against this disease is not to bring the pathogenic virus, White Spot Syndrome Virus (WSSV), to the farm. However, once the pathogenic virus enters the farm, the only way to eliminate the virus is to discard all crustaceans reared there, resulting in a significant economic loss. In addition, this pathogenic virus WSSV has a wide host range in crustaceans (Non-patent Document 1), and almost no genetic variation is observed between isolated strains (Non-patent Document 2). There is also a high risk that WSSV infection in crustaceans will spread to other species of crustaceans farmed nearby. Therefore, the development of a simple and more effective prevention method for this disease is eagerly desired.

  As a method for preventing viral diseases in crustaceans, Patent Documents 1 and 2 include WSSV structural proteins as vaccines, or live attenuated bacteria or virus vectors into which genes encoding such structural proteins have been introduced as vaccines. A method of administration to crustaceans is disclosed. However, protein vaccines are easily degraded and can elicit an immune response only for a short period of time, so they require frequent inoculations, and they require advanced techniques for production and storage, so they are disadvantageous in terms of labor and cost. . Protein vaccines can elicit a humoral immune response but it is difficult to induce a cellular immune response. On the other hand, live attenuated bacteria and viral vectors introduced with WSSV structural protein genes can elicit both humoral and cellular immune responses, but there is a risk that bacteria and viruses may recover or acquire toxicity. There is a major drawback.

  As another antiviral agent for aquatic animals, development of a DNA vaccine for fish and shellfish containing a vector encoding a viral protein as an active ingredient is also in progress (Patent Document 3). DNA vaccines are not infectious and can be maintained in vivo for long periods of time to elicit both humoral and cellular immune responses, and are easy to manufacture and store. However, with DNA vaccines, there is often a problem that an immune response cannot be elicited effectively depending on the administration method.

  In recent years, in order to enhance immunostimulation in DNA vaccines, methods have been known for incorporating unmethylated CpG motifs (unmethylated cytosine and guanine-rich DNA sequences) characteristic of bacteria into DNA vectors as adjuvants. (For example, Non-Patent Document 3 and Patent Document 3). In this method, when an unmethylated CpG motif is recognized by the receptor TLR9 and taken into the immunocompetent cell, an immune activation reaction such as production of various inflammatory cytokines is caused and the immune response is enhanced. It is considered. However, the immunostimulatory function of immunostimulatory sequences including unmethylated CpG motifs is still unclear.

Special table 2003-506338 gazette JP-T-2004-508818 Japanese Patent Laid-Open No. 9-285291 Flegel T.W., World J. Microbiol. Biotechnol. (1997) 13, p.433-442 Lo C.F. et al., Diseases of Aquatic Organisms, (1999) 35, p.175-185 Krieg A. et al., Nature, (1995) 374: p.546-549

  An object of this invention is to provide a vaccine effective in prevention of crustacean acute viremia.

As a result of intensive studies to solve the above problems, the present inventors administered a DNA vaccine prepared by incorporating the structural protein gene of WSSV virus into a pTargeT ^™ vector, which is a DNA vector for mammalian expression, to prawns. As a result, the acute viremia caused by WSSV could be prevented very effectively, and the present invention was completed based on this finding.

That is, the present invention includes the following.
[1] A DNA vaccine for preventing crustacean acute viremia containing a pTargeT ^™ vector containing the structural protein gene of white spot syndrome virus (WSSV). Structural protein genes that can be suitably used in this DNA vaccine are VP28 gene, VP26 gene, or VP19 gene.
It is particularly preferable that the crustacean to be administered in this DNA vaccine is the family Shrimp.

[2] A method for preventing crustacean acute viremia, comprising administering the DNA vaccine according to [1] above to a crustacean. In this method, the Shrimp family is particularly preferred as the crustacean to be administered.

  The DNA vaccine of the present invention can provide crustaceans with high protection against white spot syndrome virus and prevent crustacean acute viremia.

Hereinafter, the present invention will be described in detail.
The present invention relates to a DNA vaccine for preventing crustacean acute viremia comprising a pTargeT ^™ vector incorporating a structural protein gene of white spot syndrome virus (WSSV) as an active ingredient, and crustacean acute viral blood using the DNA vaccine Provide a method for preventing illness.

  Various studies have been conducted on the protein component of white spot syndrome virus (WSSV). Currently, at least 39 structural proteins (VP15, VP19, VP24, VP26, VP28, VP31, VP35, VP51C, VP36B, VP41A, VP12B) , VP73, VP180, VP664, etc.) (Jyh-Ming Tsai et al., Journal of Virology, (2006) p.3021-3029). In the DNA vaccine of the present invention, DNA encoding any structural protein can be selected as the WSSV structural protein gene. The structural protein gene is not limited, but is preferably a VP28 gene, a VP26 gene, or a VP19 gene, and more preferably a VP28 gene. In the present invention, the “structural protein gene” may be a DNA fragment isolated from WSSV genomic DNA, or may be DNA artificially produced by a recombinant method or a synthetic method. The amino acid sequence encoded by the structural protein gene according to the present invention may be a full-length sequence of the structural protein, or a partial sequence of the structural protein that is cleaved at the end but retains antigenicity. Good. The “structural protein gene” used in the DNA vaccine of the present invention may be DNA containing a known base sequence of any structural protein gene of WSSV (WSSV structural protein gene), or a known amino acid sequence of any WSSV structural protein. It may be a DNA containing a base sequence encoding. The “structural protein gene” according to the present invention consists of an amino acid sequence in which, for example, 1 to 100 (preferably 1 to 10) amino acids have been deleted, substituted or added in the known amino acid sequence of any WSSV structural protein. It may also be a DNA encoding a protein or peptide that retains its antigenicity.

  Regarding WSSV structural protein genes, many base sequences are registered in international base sequence databases and well-known base sequence databases such as GenBank. For example, but not limited to, VP15 gene: AY374120, AY249451; VP28 gene: AY324881, AY249443; VP26 gene: AY249438, AY249439; VP19 gene: AY316119, AY249444; VP24 gene: AY249457, AY249458; VP35 gene: AY325896, VP14 Gene: AY422226 etc. Regarding the VP28 gene, the open reading frame sequence determined in the Examples described later is shown in SEQ ID NO: 1, and the amino acid sequence encoded by the sequence is also shown in SEQ ID NO: 2. Alternatively, from the amino acid sequences of various WSSV structural proteins registered in a database such as GenBank, a base sequence encoding the amino acid sequence can be designed based on a known gene code.

A person skilled in the art can isolate or prepare DNA encoding each structural protein by a conventional method based on the obtained base sequences. For example, using the genomic DNA extracted from WSSV by a conventional method as a template and performing PCR using specific primer pairs designed to sandwich the target structural protein gene region from the obtained base sequence information, the target gene can be obtained. A DNA amplification product containing can be obtained. About the obtained DNA, you may introduce | transduce a variation | mutation in a base sequence by well-known site-directed mutagenesis methods, such as Kunkel method and Gapped duplex method. Site-directed mutagenesis is, for example, commercially available products such as Mutan-K ^(R) C, Mutan ^(R) -Super Express Km (Takara Bio), TAKARA LA PCR ^™ in vitro Mutagenesis kit (Takara Bio) Can also be used. The obtained DNA may be purified by any purification method known to those skilled in the art, such as an anion exchange chromatography method.

In the present invention, the WSSV structural proteins gene obtained, in pTargeT ^TM vector is a DNA vector for mammalian expression, by incorporating in the position and orientation thereto encoded protein is correctly expressed, DNA in accordance with the present invention A recombinant expression vector used as an active ingredient of a vaccine can be prepared. A mammalian expression DNA vector is a DNA vector capable of inducing expression in mammalian cells or tissues.

The pTargeT ^™ vector used in the present invention can be obtained from Promega (Madison, WI, USA). For more information on pTargeT ^TM vectors, see Promega's homepage (http://www.promega.com/vectors/tvectors.htm), or Promega's pTargeT ^TM Mammalian Expression System product documentation (this document is Also available from the following address: http://www.promega.com/tbs/tm044/tm044.html). The following summarizes the pTargeT ^™ vector information provided by Promega.

pTargeT ^TM vector
The pTargeT ^™ vector basically has a base sequence of 5670 bp in total length (shown in SEQ ID NO: 5 in the present application). The pTargeT ^™ vector is linearized by EcoRV cleavage at base number 1284 and provided with the base T added as an overhang at the 3 ′ ends of both strands (this “T” is the 5670 bp above) Not included in the sequence). Usually, using this overhang part, insert DNA can be integrated between both ends of a linearized vector. In this vector, minor mutations may be included in the above base sequence, but the restriction enzyme cleavability at the multiple cloning site is preserved.

The pTargeT ^™ vector map is shown in FIG. The components of this vector are as follows (the position of each component is based on the base number of SEQ ID NO: 5).
・ Cite Megaroyus Extreme Early Enhancer 1-659
・ Cytomegalovirus immediate early promoter 669-750
Chimeric intron 890-1022
PTargeT ^TM sequencing primer 1367-1344
LacZα start codon 1377
LacZα stop codon 1053
・ Lac operon sequence 1066-1226, 1363-1499
・ Lac operator 1397-1413
・ T7 promoter 1227-1251
・ Multicloning site 1250-1323
SV40 late polyadenylation signal 1535-1755
Phage f1 region 1798-2252
・ Neomycin selectable marker
SV40 enhancer / early promoter 2260-2630
Neomycin phosphotransferase coding region 2675-3469
Synthetic polyadenylation signal 3533-3581
β-lactamase (Amp ^r ) coding region 3978-4838

Therefore, the “pTargeT ^™ vector” in the present invention is a nucleotide sequence of SEQ ID NO: 5 or a minor mutation that does not affect vector function in the nucleotide sequence (for example, deletion of one or several (2 to 10) nucleotides, It means a DNA vector comprising a sequence in which substitution or addition has occurred.

incorporation of WSSV structural protein genes into pTargeT ^TM vector according to the instructions of Promega Corporation pTargeT ^TM Mammalian Expression System, performed by ligation reaction with the insert DNA containing the pTargeT ^TM vector and WSSV structural protein gene linearized be able to.

A chimeric plasmid DNA (expression vector) in which the WSSV structural protein gene according to the present invention is incorporated into a pTargeT ^™ vector can continuously express the WSSV structural protein gene in vivo and in vitro. The chimeric plasmid DNA is also taken up and retained in the cell. Therefore, when the chimeric plasmid DNA according to the present invention is administered in vivo, WSSV structural protein is continuously produced, and humoral immune response (antibody production) and cellular immune response against WSSV are induced over a long period of time. Furthermore, this chimeric plasmid DNA according to the present invention itself has a strong immunostimulatory function by using the pTargeT ^™ vector as the backbone sequence.

  In the present invention, such a chimeric plasmid DNA according to the present invention can be used as an active ingredient of a DNA vaccine for preventing crustacean acute viremia caused by WSSV infection. In this case, the chimeric plasmid DNA as an active ingredient is more preferably isolated and purified by any purification method known to those skilled in the art, such as an anion exchange chromatography method.

In the present invention, “DNA vaccine” means a vaccine preparation comprising as an active ingredient a DNA expression vector incorporating a DNA encoding an antigen protein or antigen peptide. The DNA vaccine in the present invention is a vaccine in which a pharmaceutically acceptable carrier (which can be added to a pharmaceutical product) or an additive is added in addition to an effective amount of a chimeric plasmid DNA in which a WSSV structural protein gene is incorporated into a pTargeT ^™ vector. It may be a composition. Carriers and additives include water, isotonic solutions, aqueous buffers, pharmaceutically acceptable organic solvents and surfactants, auxiliaries, stabilizers, antioxidants, preservatives, and the like. More specific examples of carriers and additives include, but are not limited to, sterile water, saline, Ringer's solution, PBS, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, Sodium carboxymethyl starch, pectin, xanthan gum, gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose, and other liposomes Artificial cell structures such as These carriers and additives are appropriately selected according to the administration route, dosage form, storage form and the like of the DNA vaccine. The DNA vaccine of the present invention may further contain an adjuvant, but since it originally has a high immunostimulatory function, it is not always necessary to add an additional adjuvant. The DNA vaccine of the present invention may further contain other pharmacological components.

  The DNA vaccine according to the present invention exhibits a high effect on the prevention of crustacean acute viremia when administered to crustaceans. In the present invention, “crustacean acute viremia” means a crustacean infection caused by white spot syndrome virus (WSSV) (also called white spot syndrome, leukosis etc.). This disease is typically characterized by white spots appearing on the body surface and death at an extremely high rate. In the present invention, “prevention of crustacean acute viremia” means that WSSV that has entered the living body is excluded and / or its growth is suppressed, and as a result, the development of crustacean acute viremia is prevented or It means to reduce the incidence significantly. The preventive effect of crustacean acute viremia obtained by administration (vaccination) of the DNA vaccine of the present invention is typically indicated by an increase in survival rate (decrease in mortality rate) in the presence of WSSV.

  In the present invention, the crustacea to be administered with the DNA vaccine may be any crustacea that can be infected with WSSV, such as shrimp, crab, crayfish, crayfish, krill, daphnia, etc. Is mentioned. Particularly suitable examples of these crustaceans include, but are not limited to, decapod crustaceans, such as the shrimps, shrimps, lobsters, kingfishes, red lobsters, lobsters, lobsters, crayfishes And other organisms belonging to the genus, such as the Decapoda crabs, spider crabs, crabs, crabs, crabs, and crabs. More specific examples of crustaceans suitable for administration include, but are not limited to, organisms in the family Penaeidae, such as Farfantepenaeus, Fenneropenaeus, Litopenaeus, Marsupenaeus, Melicertus, Metapenaeopsis, Metapenaeus, Penacheus, Examples include shrimp belonging to the genus Xiphopenaeus. Among the shrimps that are suitable for administration, for example, edible shrimp include shrimp (Marsupenaeus japonicus), southern shrimp (Melicertus canaliculatus), shrimp (black tiger) (Penaeus monodon), red shrimp (Penaeus chinensis), bear shrimp (Penaeus semi) ), Yellow shrimp (Penaeus latisulcatus), Indian shrimp (Fenneropenaeus indicus), reed shrimp (Metapenaeus ensis), horse shrimp (Metapenaeus intermedius) and the like, but are not limited thereto.

  The DNA vaccine according to the present invention can be administered to crustaceans by any appropriate administration method. The DNA vaccine of the present invention is not limited, but can be administered to crustaceans, for example, by injection, immersion, spraying, or orally. More preferably, the DNA vaccine of the present invention can be administered to crustaceans by intramuscular, intraperitoneal, etc. injection. The dose of the DNA vaccine is preferably 0.1 to 50 μg, more preferably 1 to 10 μg, per animal to be administered. The DNA vaccine may be administered only once, but may be repeated two or more times at intervals.

By administering the DNA vaccine according to the present invention, the expression of the WSSV structural protein gene is induced from the chimeric plasmid DNA, which is the active ingredient of the DNA vaccine, in the crustacean body, and as a result, the WSSV structural protein is produced. Both humoral and cellular immune responses are elicited. At the same time, the chimeric plasmid DNA itself exerts an adjuvant function and brings about a high immunostimulatory effect. In the present invention, the use of the pTargeT ^™ vector for the production of chimeric plasmid DNA has succeeded in obtaining a particularly excellent immunostimulatory effect. According to the administration of the DNA vaccine of the present invention, crustacean acute viremia can be very effectively prevented by the induction of such an immune response and the remarkable immune activation effect.

  The DNA vaccine of the present invention has a significantly higher immunity enhancing effect than conventional vaccines that use viral proteins, attenuated viruses, modified viruses and the like as vaccine antigens. In addition, the DNA vaccine of the present invention is much easier to manufacture than those of the conventional vaccines, can be stably stored for a long period of time, has no risk of infection, and can be further modified. In addition, there is little risk of contaminating the aquatic environment when administering the DNA vaccine of the present invention. Accordingly, the method for preventing crustacean acute viremia by administering the DNA vaccine of the present invention can be used very advantageously in, for example, the crustacean aquaculture industry.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

[Example 1] Preparation of culture medium and buffers The culture medium and buffer used in Examples described later were prepared as follows.
a) SOC medium Each component was mixed according to the following composition, autoclaved, cooled to 60 ° C. or lower, and then added with 20 ml of 1M glucose to prepare a medium. Immediately before use, 100 μl each of 1M MgCl ₂ and 1M MgSO ₄ sterilized by filtration per 1 ml of the medium was added to this medium, and used as the SOC medium in the following examples.
SOC medium
Bacttripton 20g
Bact yeast extract 5g
NaCl 0.5g
Add distilled water to adjust to 1L

b) 1.6g AX plate bacto tryptone
Bact yeast extract 1.0g
NaCl 0.5g
Agar 1.5g
Add distilled water to adjust to 100 ml

Each component was mixed and autoclaved according to the above composition. Then, after cooling to 40-50 degreeC, the following reagent was added to the culture medium in the following amounts.
Ampicillin (50 mg / ml) 100 μl
X-gal (200 mg / ml) 100 μl
20% IPTG 10μl
The medium to which the reagent was added was mixed well, then dispensed into a petri dish (20 ml / plate), cooled and hardened to obtain an AX plate to be used in a later example.

c) PBS
Each component was mixed according to the following composition, and the pH adjusted to 7.6 was autoclaved. The PBS thus prepared was used in a later example.
PBS
NaCl 28.4 g
MgCl ₂・ 6H ₂ O 1.0 g
MgSO ₄・ 7H ₂ O 2.0 g
CaCl ₂・ 2H ₂ O 2.25 g
KCl 0.7 g
Glucose 1.0 g
HEPES 2.38 g
Add distilled water to adjust to 1L

[Example 2] Cloning of WSSV-VP28 gene A shark of Marsupenaeus japonicus infected with White Spot Syndrome Virus (WSSV) was extracted and then DNeasy Tissue Kit (QIAGEN, USA) was used. Genomic DNA was extracted. Using the extracted genomic DNA as a template, a primer pair specific to the WSSV envelope protein VP28 gene [forward primer VP28-Fw 5'-ATGGATCTTTCTTTCAC-3 ', reverse primer VP28-Rv 5'-TTACTCGGTCTCAGTGC-3'] PCR was performed. PCR reaction solution is dNTP mixture (dATP, dCTP, dGTP, dTTP 2.5 mM each) and 10 × Gene Taq Universal Buffer 5 μl each, Taq polymerase (5 U / μl) (Nippon Gene, Japan) 0.5 μl, 5 μl each of 2.5 μM VP28-Fw primer and VP28-Rv primer, 28.5 μl of sterilized distilled water, and 1 μl of genomic DNA were mixed and used as a total of 50 μl. PCR conditions were as follows: heat denaturation at 94 ° C for 3 minutes, 94 cycles for 30 seconds, 60 ° C for 30 seconds, 72 ° C for 1 minute, 30 cycles, and then 72 ° C for 5 minutes. An extension reaction was performed. The size (about 615 bp) of the amplified product was confirmed by electrophoresis using a 1.5% agarose gel. The amplification product thus obtained is a DNA fragment containing the WSSV-VP28 gene.

In principle, the WSSV-VP28 gene was cloned according to the instruction manual of pTargeT ^™ Mammalian Expression Vector System (PROMEGA, USA). First, 1 μl of the PCR product specifically amplified as described above was mixed with 1 μl of 10 × ligation buffer, 1 μl of pTargeT ^™ vector and 1 μl of 3 U / μl T4 DNA ligase (both PROMEGA, USA). The WSSV-VP28 gene fragment was incorporated into a pTargeT ^™ vector by performing a ligation reaction by allowing to stand overnight at ° C. Subsequently, in order to transform Escherichia coli with the obtained WSSV-VP28 gene fragment-containing vector, 1 μl of the ligation product was added to 50 μl of E. coli competent cell JM109 (Nippon Gene, Japan) and then allowed to stand on ice for 30 minutes. . The E. coli after standing was subjected to heat shock at 42 ° C. for 45 seconds and further left on ice for 2 minutes. After this standing, 500 μl of SOC medium was added, followed by shaking culture (200 rpm / min) in a 37 ° C. water bath for 1 hour 30 minutes. 100 μl of the obtained culture was added onto an AX plate, and spread over the entire surface using a large rod. Leave overnight at 37 ° C, select a clone (white colony) containing the target insert DNA by blue-white selection, use the QIAprep Spin Miniprep Kit (Qiagen, USA) from this clone, and plasmid according to the kit manual. DNA extraction was performed. Confirmation of the base sequence of the insert DNA was performed by sequence analysis with a sequencer LIC-4200L (Li-Cor, USA) using a T7 primer. The insert DNA contained the ORF sequence of the VP28 gene shown in SEQ ID NO: 1. The nucleotide sequence of the VP28 gene in the insert DNA thus analyzed is the VP28 gene sequence of many WSSV overseas isolates registered on the international nucleotide sequence database [Indian isolates: AY873785, DQ013881, DQ013882, DQ681069. , DQ902658, Dutch isolate: AF173993, Taiwan isolate: AF272979, Vietnam isolate: AY168644, AJ551447, China isolate: DQ007315, AF227911, AY249440, DQ098011, Indonesia isolate: AY249441, US isolate: AY249442, Japan isolate : AY249443, Korean isolate: AY324881; both showed accession number] and showed 100% sequence homology. The amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 2.

The plasmid DNA obtained as described above is a chimeric plasmid DNA in which the WSSV-VP28 gene is incorporated downstream of the CMV (cytomegalovirus) promoter in the pTargeT ^™ vector. Among the obtained chimeric plasmid DNAs, the insert DNA (WSSV-VP28 gene fragment) in which the correct sequence was confirmed and good amplification was confirmed was named pCMV-VP28.

[Example 3] Infection experiment (dipping method)
In this example, WSSV infection was carried out using the chimeric plasmid DNA (pCMV-VP28) prepared in Example 2 and incorporated into the pTargeT ^™ vector in a state in which expression can be induced in vivo. DNA vaccination was carried out against the marine prawn (Marsupenaeus japonicus) and the effect was judged (FIG. 1).

  First, pCMV-VP28 was prepared as a 100 μg / ml PBS solution to be used as a vaccine plasmid DNA solution for injection. For the WSSV for infection experiments, the heart, sputum, stomach and midgut glands were removed from 3 tailed prawns infected with WSSV, added with 2 ml of PBS and homogenized, and further added with PBS to a total volume of 30 ml. Prepared as the resulting solution (WSSV solution).

  Subsequently, 100 μg / ml PBS solution of pCMV-VP28 prepared as described above was administered as a vaccine plasmid DNA solution to a group of prawns having an average body weight of 8.0 g by injection into the shrimp muscles after injection. They were raised for 1 week in a circulating water tank (60 x 30 x 35 cm) with a water temperature of 24 ° C. Next, move the prawns into a tank containing 2 L of artificial seawater, add 5 ml of the WSSV solution prepared above to the artificial seawater, suspend it, and perform aeration attack on the prawns for 2 hours while performing sufficient aeration. Was done. After the attack, death shrimp were counted over time to determine the cumulative mortality rate and create a graph (Fig. 2). As a control experiment, a prawn infection experiment was performed in the same procedure except that PBS was inoculated instead of the vaccine plasmid DNA solution.

  As a result, in the first infection experiment (FIG. 2A), significant death began to be confirmed on the third day after the start of the infection experiment. The control (PBS inoculation) group continued to die, and at the end of the infection test, the cumulative mortality rate was 71.5% (28.5% survival rate). On the other hand, in the pCMV-VP28 vaccination group, no deaths were observed after the third day, and the cumulative mortality rate at the end of the study (day 12) was 21.5% (78.5% survival rate), compared with the control group It was very low. The vaccinated group showed a significantly lower cumulative mortality rate than the control group throughout the study period after death began.

  In the second infection experiment (Fig. 2B) conducted in the same way, significant death began to be confirmed on the second day after the start of the infection experiment, and until the end of the test (day 15) in both the control group and the pCMV-VP28 vaccination group Death continued. However, the cumulative mortality rate at the end of the study was 93% in the control group (survival rate 7%), whereas the pCMV-VP28 vaccinated group was 47% (survival rate 53%), which was much higher than the control group. It was low. In this second infection experiment, the pCMV-VP28 vaccinated group showed a significantly lower cumulative mortality rate than the control group throughout the test period after the start of death.

  The DNA vaccine of the present invention can be used as an effective preventive measure against crustacean viremia that causes enormous damage in the aquaculture industry.

FIG. 1 is a diagram showing an outline of a WSSV infection experiment for determining the effect of DNA vaccination. FIG. 2 is a diagram showing the effect of DNA vaccination in WSSV infection experiments. 2A and 2B are graphs showing changes over time in survival rates in infection experiments conducted at the first time and the second time, respectively.

  The sequences of SEQ ID NOs: 3 and 4 are primers.

The sequence of SEQ ID NO: 5 is a pTargeT ^™ vector.

Claims (5)

A DNA vaccine for preventing crustacean acute viremia containing a pTargeT ^™ vector containing the structural protein gene of white spot syndrome virus (WSSV).   The DNA vaccine according to claim 1, wherein the structural protein gene is a VP28 gene, a VP26 gene, or a VP19 gene.   The DNA vaccine according to claim 1 or 2, wherein the crustacean is a family of prawns.   A method for preventing crustacean acute viremia, comprising administering the DNA vaccine according to any one of claims 1 to 3 to a crustacean.   The method according to claim 4, wherein the crustacean is a prawn family.

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